Autoimmunity
Abstract
Autoimmune diabetes encompasses rapidly progressive type 1 diabetes mellitus (T1D) and indolent latent autoimmune diabetes in adults (LADA), representing distinct inflammatory set points along a shared autoimmune spectrum. Yet the immunological mechanisms that determine these divergent inflammatory states remain unresolved. We performed single-cell RNA sequencing with paired T and B cell receptor profiling on over 400,000 peripheral blood mononuclear cells (PBMCs) from patients with LADA, newly diagnosed T1D, and healthy controls. PBMC composition was comparable across cohorts, indicating that qualitative rather than quantitative immune differences underlie disease heterogeneity. In T1D, pan-lineage activation of NF-κB, EGFR, MAPK, and hypoxia pathways, coupled with a TNF-centered communication hub, enhanced MHC signaling, and disrupted adhesion, promoted systemic inflammation. LADA, by contrast, exhibited global suppression of NF-κB/EGFR activity, retention of moderate JAK/STAT tone, reinforced natural killer cell inhibitory checkpoints via HLA-C–KIR2DL3/3DL1 interaction, and stabilized CD8⁺ T cell synapses through HLA-C–CD8 binding, collectively restraining effector activation. Single-cell V(D)J analysis revealed multiclonal, patient-unique adaptive repertoires, emphasizing the primacy of signaling context over receptor convergence. These findings define autoimmune diabetes as an inflammatory–inhibitory set-point continuum, positioning the NF-κB/EGFR–JAK/STAT gradient and HLA-C–KIR axis as potential therapeutic targets to preserve residual β-cell function.
Authors
Ivan I. Golodnikov, Elizaveta S. Podshivalova, Vadim I. Chechekhin, Anatoliy V. Zubritskiy, Alina A. Matrosova, Nikita A. Sergeev, Margarita D. Samsonova, Yaroslav V. Dvoryanchikov, Tatiana V. Nikonova, Ekaterina V. Bondarenko, Marina Yu. Loguinova, Yulia A. Medvedeva, Dmitry N. Laptev, Rita I. Khusainova, Ildar R. Minniakhmetov, Marina V. Shestakova, Natalia G. Mokrysheva, Ivan I. Dedov
Abstract
The two main subgroups of autoimmune myasthenia gravis, a neuromuscular junction disorder associated with muscle weakness, are the early and late-onset forms, defined by onset before or after 50 years of age. Both carry acetylcholine-receptor autoantibodies, but differ in sex ratios, genetics and occurrence of disease-specific thymus inflammation. By applying multimodal techniques, including deep spectral cytometric phenotyping and single cell sequencing to peripheral blood and thymic lymphocyte samples we explored the possibility to discriminate the two forms by cellular immune phenotyping. Analyzing two independent cohorts we identified distinct immunological differences driven by three main lymphocyte populations. Lower frequencies of mucosa-associated invariant T cells and naïve CD8 T cells were observed in late-onset myasthenia, suggesting enhanced immune senescence. Further, a highly differentiated, canonical natural killer cell population was reduced in early-onset myasthenia, which was negatively correlated with the degree of thymic inflammation. Using only the frequency of these three populations, correct myasthenia subgroup assignment could be predicted with an accuracy of 90%. The NK cell population negatively associated to early-onset disease had a similar association to thymic hyperlasia, whereas the two T-cell populations point to enhanced immune senescence in late-onset myasthenia gravis. These distinct immunocellular endophenotypes for early- and late onset disease suggest differences in the immunopathogenic processes. Together with demographic factors and other disease subgroup-specific features, the frequency of the identified cell subpopulations may improve clinical classification, in turn of relevance for channeling to interventions.
Authors
Jakob Theorell, Nicolas Ruffin, Andrew Fower, Chiara Sorini, Philip Ambrose, Valentina Damato, Lahiru Handunnetthi, Isabel Leite, Sarosh R. Irani, Susanna Brauner, Adam E. Handel, Fredrik Piehl
Abstract
Immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-CTLA-4 antibodies are used to induce an immune response against many types of tumors. However, ICIs often also induce autoimmune responses, referred to as immune-related adverse events (irAEs), which occur unpredictably and at varying levels of severity in ICI-treated patients. The immunologic factors that predispose patients to the development of severe irAE are largely unclear. Here, we utilized high dimensional mass cytometry immunophenotyping of longitudinal blood samples from patients with metastatic melanoma treated with combination anti-PD-1/CTLA4 ICI therapy in the context of a clinical trial to characterize alterations in immune profiles induced by combination ICI therapy and to identify immune features associated with development of severe irAEs. Deep T cell profiling highlighted that ICI therapy induces prominent expansions of activated, CD38hi CD4+ and CD8+ T cells, which are frequently bound by the therapeutic anti-PD-1 antibody, as well as substantial changes in regulatory T cell phenotypes. However, neither the baseline frequency nor the extent of expansion of these cell populations was associated with development of severe irAEs. Rather, single cell-association testing revealed naïve CD4+ T cell abundance pre-treatment as significantly associated with the development of severe irAEs. Biaxial gating of naïve CD4+ T cells confirmed a significant positive association of naïve CD4+ T cell proportion and development of a severe irAE and with the number of irAEs developed in this cohort. Results from this broad profiling study indicate the abundance of naïve CD4+ T cells as a predictive feature for the development of severe irAEs following combination anti-PD-1/CTLA4 ICI therapy.
Authors
Kathryne E. Marks, Alice Horisberger, Mehreen Elahee, Ifeoluwakiisi A. Adejoorin, Nilasha Ghosh, Michael A. Postow, Laura Donlin, Anne R. Bass, Deepak A. Rao
Abstract
BACKGROUND. Individuals with autoimmune diseases (AD) on immunosuppressants often have suboptimal responses to COVID-19 vaccine. We evaluated the efficacy and safety of additional COVID-19 vaccines in those treated with mycophenolate mofetil/mycophenolic acid (MMF/MPA), methotrexate (MTX), and B cell-depleting therapy (BCDT), including the impact of withholding MMF/MPA and MTX. METHODS. In this open-label, multicenter, randomized trial, 22 participants taking MMF/MPA, 26 taking MTX, and 93 treated with BCDT who had suboptimal antibody responses to initial COVID-19 vaccines (2 doses of BNT162b2 or mRNA-1273 or 1 dose of AD26.COV2.S) received an additional homologous vaccine. Participants taking MMF/MPA and MTX were randomized (1:1) to continue or withhold treatment around vaccination. The primary outcome was the change in anti-Wuhan-Hu-1 receptor-binding domain (RBD) concentrations at 4 weeks post-additional vaccination. Secondary outcomes included adverse events, COVID-19 , and AD activity through 48 weeks. RESULTS. Additional vaccination increased anti-RBD concentrations in participants taking MMF/MPA and MTX , irrespective of immunosuppressant withholding. BCDT-treated participants also demonstrated increased anti-RBD concentrations, albeit lower than MMF/MPA- and MTX-treated cohorts. COVID-19 occurred in 33% of participants; infections were predominantly mild and included only three non-fatal hospitalizations. Additional vaccination was well-tolerated, with low frequencies of severe disease flares and adverse events. CONCLUSION. Additional COVID-19 vaccination is effective and safe in individuals with ADs treated with immunosuppressants, regardless of whether MMF/MPA or MTX is withheld. TRIAL REGISTRATION. ClinicalTrials.gov (NCT05000216; registered August 6, 2021: https://clinicaltrials.gov/ct2/show/NCT05000216)
Authors
Meggan Mackay, Catriona A. Wagner, Ashley Pinckney, Jeffrey A. Cohen, Zachary S. Wallace, Arezou Khosroshahi, Jeffrey A. Sparks, Sandra Lord, Amit Saxena, Roberto Caricchio, Alfred H.J. Kim, Diane L. Kamen, Fotios Koumpouras, Anca D. Askanase, Kenneth Smith, Joel M. Guthridge, Gabriel Pardo, Yang Mao-Draayer, Susan Macwana, Sean McCarthy, Matthew A. Sherman, Sanaz Daneshfar Hamrah, Maria Veri, Sarah Walker, Kate York, Sara K. Tedeschi, Jennifer Wang, Gabrielle E. Dziubla, Mike Castro, Robin Carroll, Sandeep R. Narpala, Bob C. Lin, Leonid Serebryannyy, Adrian B. McDermott, William T. Barry, Ellen Goldmuntz, James McNamara, Aimee S. Payne, Amit Bar-Or, Dinesh Khanna, Judith A. James
Abstract
Peripheral helper T (Tph) and follicular helper T (Tfh) cells are key regulators of B cell differentiation and antibody production, making them promising targets for autoimmune disease treatment. However, their differentiation mechanisms differ significantly between humans and mice, limiting drug validation in mouse models. Here, we present a simple and effective method for in vivo proliferation of human Tph/Tfh and B cells. We discovered that after depleting CD8+ T cells of human peripheral blood mononuclear cell–transferred immunodeficient mice (CD8TΔhPBMC mice), human Tph/Tfh cells and B cells proliferated markedly in the spleen compared with those in human PBMC–transferred immunodeficient mice (hPBMC mice). Transcriptome analysis confirmed proliferating cells’ close resemblance to human Tph/Tfh cells. Furthermore, multicolor flow cytometry revealed CXCL13+ Tph cells infiltrating Sjögren’s syndrome–associated (SjS-associated) organs, such as salivary glands. Single-cell RNA sequencing identified IL-21+CXCL13+IFN-γ+ICOS+TIGIT+GPR56+ Tph cells in the salivary glands. These findings are consistent with reduced saliva volume and elevated SjS markers, such as anti-SSA antibody, in these mice, which were both ameliorated by immunosuppressants. In vitro, CD8+ T cells from hPBMC mice induced B cell apoptosis and inhibited Tph/Tfh differentiation. This model advances understanding of human Tph/Tfh cell biology and offers a valuable platform for studying SjS and therapeutic targets.
Authors
Mariam Piruzyan, Sota Fujimori, Ryota Sato, Yuki Imura, Sachiko Mochiduki, Kana Takemoto, Akiko Nishidate, Yuzo Koda
Abstract
Juvenile idiopathic arthritis (JIA) is the most prevalent chronic inflammatory arthritis of childhood, yet the spatial organization in the synovium remains poorly understood. Here, we perform subcellular-resolution spatial transcriptomic profiling of synovial tissue from patients with active JIA. We identify diverse immune and stromal cell populations and reconstruct spatially defined cellular niches. Applying a newly developed spatial colocalization analysis pipeline, we uncover microanatomical structures, including endothelial–fibroblast interactions mediated by NOTCH signalling, and a CXCL9-CXCR3 signaling axis between inflammatory macrophages and CD8+ T cells, alongside the characterization of other resident macrophage subsets. We also detect and characterize tertiary lymphoid structures marked by CXCL13-CXCR5 and CCL19-mediated signaling from Tph cells and immunoregulatory dendritic cells, analogous to those observed in other autoimmune diseases. Finally, comparative analysis with rheumatoid arthritis reveals JIA-enriched cell states, including NOTCH3+ and CXCL12+ sublining fibroblasts, suggesting potentially differential inflammatory programs in pediatric versus adult arthritis. These findings provide a spatially resolved molecular framework of JIA synovitis and introduce a generalizable computational pipeline for spatial colocalization analysis in tissue inflammation.
Authors
Jun Inamo, Roselyn Fierkens, Michael R. Clay, Anna Helena Jonsson, Clara Lin, Kari Hayes, Nathan D. Rogers, Heather Leach, Kentaro Yomogida
Abstract
Acute rheumatic fever (ARF) and associated rheumatic heart disease are serious sequelae of a Group A Streptococcus (GAS/Strep A) infection. Autoantibodies are thought to contribute to pathogenesis, with deeper exploration of the autoantibody repertoire needed to improve mechanistic understanding and identify new biomarkers. Phage immunoprecipitation and Sequencing (PhIP-Seq) with the HuScan library (>250,000 overlapping 90-mer peptides spanning the human proteome) was utilised to analyse autoreactivity in sera from children with ARF, uncomplicated Strep A pharyngitis and matched healthy controls. A global proteome-wide increase in autoantigen reactivity was observed in ARF, as was marked heterogeneity between patients. Public epitopes, common between individuals with ARF were rare, and comprised < 1% of all enriched peptides. Differential analysis identified both novel and previously identified ARF autoantigens, including PPP1R12B, a myosin phosphatase complex regulatory subunit expressed in cardiac muscle, and members of the collagen-protein family, respectively. Pathway analysis found antigens from the disease-relevant processes encompassing sarcomere and heart-morphogenesis were targeted. In sum, PhIP-Seq has substantially expanded the spectrum of autoantigens in ARF, and reveals the rarity of public epitopes in the disease. It provides further support for the role of epitope spreading in pathogenesis and has identified PPP1R12B as a novel, enriched autoantigen.
Authors
Reuben McGregor, Lauren H. Carlton, Timothy J. O'Donnell, Elliot Merritt, Campbell R. Sheen, Florina Chan Mow, William John Martin, Michael G. Baker, Nigel Wilson, Uri Laserson, Nicole J. Moreland
Abstract
The efficacy of anti-CD20 therapies places B cells and their interaction with T cells at the center of attention for multiple sclerosis (MS) pathogenesis. Follicular T helper cells (Tfh), which guide B cell maturation in germinal centers within lymph nodes (LNs), are elevated in the circulation and cerebrospinal fluid of patients with MS (pwMS). However, the LN spatial landscape has remained largely without investigation for pwMS. Using cyclic immunofluorescence, we assessed cell abundance and spatial connections in FFPE LNs of 33 pwMS and 35 non-MS controls. The presence of EBV was analyzed through EBER immunostaining and multiplex quantitative PCR. Our analysis showed that Tfh cells were expanded in LNs of pwMS and accumulated especially in the mantle zone and B cell follicles compared with controls. The Tfh/T follicular regulator ratio was increased in pwMS, while B cell ratios were similar between the cohorts. The interaction of Tfh cells with follicular B cells was higher in pwMS. Interestingly, Tfh accumulation was also observed in 5 prediagnostic MS cases. No signs of EBV latency were detected in either group. These findings highlight LNs as a site of early and persistent immune activation in pwMS, with therapeutic implications to be further addressed.
Authors
Joona Sarkkinen, Eliisa Kekäläinen, Leo Hannolainen, Ada Junquera, Johannes Dunkel, Maria F. Perdomo, Mikko I. Mäyränpää, Sini M. Laakso
Abstract
In the rheumatoid arthritis (RA) synovium, resident fibroblast-like synoviocytes (FLS) express MHC class II molecules (HLA-D) but lack the co-stimulatory signals typically required for T cell activation. Here, we demonstrate that antigen presentation by FLS induces a distinct T cell activation state characterized by high CD69, yet reduced CD25 and HLA-DR expression, suppressed proliferation, and decreased effector cytokine production compared to professional antigen presenting cells (APCs), such as macrophages. FLS were also capable of suppressing macrophage-induced T cell activation, underscoring their dominant immunomodulatory role in the synovial microenvironment. Mechanistically, we identify indoleamine 2,3-dioxygenase (IDO1)-mediated tryptophan depletion as the primary driver of FLS-induced T cell hypo-responsiveness. Spatial transcriptomics revealed colocalization of IDO1 and CD69 within ectopic lymphoid structures in RA synovium, further supporting the in vivo relevance of this pathway. These findings provide the groundwork for positioning FLS as critical T cell regulators in RA and highlight the importance of preserving their immunosuppressive properties when therapeutically targeting pathogenic FLS functions.
Authors
Melissa R. Romoff, Preethi K. Periyakoil, Edward F. DiCarlo, Daniel Ramirez, Susan M. Goodman, Christina S. Leslie, Alexander Y. Rudensky, Laura T. Donlin, Melanie H. Smith
Abstract
Rheumatoid arthritis (RA) is a common systemic autoimmune disorder. Fibroblast-like synoviocytes (FLS) have emerged as an attractive target for non-immunosuppressive RA therapy, but there are no approved drugs targeting FLS. The receptor protein tyrosine phosphatase sigma (PTPRS) negatively regulates FLS migration and has been proposed as a target for FLS-directed RA therapy. Here we examined the impact of sequence variations on efficacy of an FLS-targeted biologic comprised of Fc-fused PTPRS immunoglobulin (IgG)-like domains Ig1 and Ig2 (Ig1&2-Fc). Engineering the linker and Fc tag improved effectiveness of human Ig1&2-Fc in assays of FLS migration and a mouse model of arthritis. Treatment of mice with Ig1&2-Fc over four months revealed no signs of toxicity or organ pathology. Finally, we show potential of Ig1&2-Fc co-administration in combination or as a bispecific fusion with a tumor necrosis factor alpha inhibitor (TNFi). Combination treatment of mouse tumor necrosis factor receptor 2 (mTnfr2) with Ig1&2-Fc resulted in increased efficacy in suppressing arthritis beyond single agent treatment. When administered as a dual-action bispecific, Ig1&2 fused to mTnfr2 proved more efficacious at suppressing arthritis than mTnfr2 alone. This study illustrates the potential of Ig1&2-Fc as a combination or bispecific therapy with DMARDs to improve patient outcomes in RA.
Authors
Sterling H. Ramsey, Zixuan Zhao, Megan C. Lee, Thales Hein da Rosa, Ava C. Schneider, Miriam Bollmann, Nour Dada, Katie Frizzi, May M. Han, Jaeyeon Kim, Martina Zoccheddu, Nigel A. Calcutt, Gary S. Firestein, James W. Bryson, Mattias N.D. Svensson, Eugenio Santelli, Stephanie M. Stanford, Nunzio Bottini
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